Process for the production of 2-halo-6-aminopurine derivatives

ABSTRACT

A novel process for preparing 2-halo-6-aminopurine derivatives and their analogs is disclosed. The method comprises halogenation of 2,6-diaminopurine derivatives at the C-2 position in a specific combination of aprotic polar and nonpolar organic solvents to give the corresponding halogenated derivatives.

This application is a continuation of application Ser. No. 09/274,518filed Mar. 23, 1999, now U.S. Pat. No. 6,252,061. This applicationclaims priority under 35 U.S.C. §119 from provisional application Ser.No. 60/079,059, filed Mar. 23, 1998, the disclosure of which isincorporated herein in its entirety.

FIELD OF THE INVENTION

The invention relates to a novel process for preparing2-halo-6-aminopurine compounds and derivatives thereof, comprisinghalogenation of 2,6-diaminopurine compounds at the C-2 position to givethe corresponding halogenated compounds, including halogenated

nucleosides. These nucleosides, e.g.,2-chloro-6-aminopurine-2′-deoxyribonucleoside, i.e., the compound withthe above structure wherein R is 2′-deoxyribose and X is Cl(2-chloro-2′-deoxyadenosine), are useful as antileukemic agents, e.g.,in treating leukemias such as hairy cell leukemia.

The invention also relates to methods for the synthesis of acyclicderivatives of 2-halo-6-aminopurines and 2-halo-6-aminopurine morpholinoderivatives which are useful in the preparation of syntheticoligonucleotide analogs.

BACKGROUND OF THE INVENTION

Processes for preparing 2-chloro-6-aminopurine-2′-deoxyribonucleoside(2-chloro-2′-deoxyadenosine, hereinafter “2-CdA”) and other2-chloro-6-aminopurines are known in the art. Such processes aredescribed in, e.g., U.S. Pat. No. 4,760,137; Kazimierzuk et al., J. Am.Chem. Soc., 106:6379, 1984; Wright et al., J. Org. Chem., 52:4617, 1987and Christensen et al., J. Med. Chem., 15:735, 1972. The preparation of2-CdA described by these workers requires the glycosylation of adihalogenated purine to give an intermediate dihalogenated nucleosidewhich is then transformed into the desired nucleoside. Morespecifically, these workers described the glycosylation of2,6-dichloropurine with1-chloro-2′-deoxy-3′,5′-di-O-p-toluyl-β-D-erythropentofuranose to give amixture of N-7 and N-9 isomers of2,6-dichloro-(2′-deoxy-3′,5′-di-O-p-toluyl-β-D-erythropentofuranosyl)-purine.This process suffers from several shortcomings, such as the formation ofisomeric side products at the 1′-carbon and the utilization of costlystarting materials, such as 2,6-dichloropurine.

U.S. Pat. No. 5,208,327 discloses a method for preparation of 2-CdA fromguanosine in eight steps via a 2-chloroadenosine intermediate in 2.8%overall yield (from guanosine). This method is inefficient and requiresseveral protection and deprotection steps in order to remove the 2′hydroxyl to yield a 2′-deoxy product. The synthesis of the2-chloroadenosine intermediate also disclosed in the same patent usesprotecting group chemistry and an alternate halogenation/aminationstrategy. This process is extremely expensive because of the multiplesteps involved and the use of expensive 2-chloroguanosine startingmaterial, and is not suitable for truly large scale production.

Processes for the preparation of compounds of the formula:

wherein R¹ is acyl or tolyl and W¹ and W² are independently halogen oramino from the corresponding per-O-protected nucleosides are disclosed,e.g., in Robins & Uznanski, Can. J. Chem. 59, 2601, 1981; Montgomery &Hewson, J. Med. Chem. 12, 498, 1969; and Huang et. al. J. Med. Chem.,27, 800-802, 1984. The transformation of the starting nucleosides to2-halopurines requires several steps, including diazotization of the2-amino intermediates in non-polar organic solvents, followed byhalogenation. Thus, this method is completely unfeasible when it isnecessary to utilize starting materials that are not soluble or onlysparingly soluble in non-polar organic solvents, in contrast to themethods of the present invention, detailed below.

Methods for the conversion of unprotected purine ribonucleosides havingthe formula:

wherein R¹ is hydrogen, W³ is halogen or hydrogen, and W¹ and W² areindependently amino or halogen, to 2-halogenated nucleosides are knownin the art (Gerster et. al., J. Org. Chem., 33, 1070, 1968; Gerster et.al., J. Org. Chem., 31, 3258, 1966; Gerster et. al., J. Am. Chem. Soc,87, 3752, 1965). However, these methods provide low yields of products,and require reactions to be performed with sodium nitrite attemperatures below 0° C. in aqueous solution, thus making drying andseparation of products difficult. The prior art also discloses thatdiazotization of 2-amino groups is only possible for ribonucleosides,because the reaction conditions cleave the glycosyl linkage of thecorresponding deoxynucleosides (see Montgomery & Hewson, J. Med. Chem.12, 498, 1969).

Thus, while the prior art discloses processes for the preparation of2-CdA and other 2-halo-6-amino nucleosides and deoxynucleosides, thesemethods all have disadvantages, such as including a glycosylationreaction, or the need for a series of nucleoside hydroxylprotection/deprotection reactions, or the need to manipulate2-halo-ribonucleosides or analogs at sub-zero temperatures using aqueousreaction conditions.

The present inventors have now surprisingly and unexpectedly discoveredmethods that make it possible to convert unprotected 2′- or3′-deoxynucleosides, ribonucleosides or analogs to the corresponding2-halo derivatives. Also discovered by the present inventors are methodsfor performing such transformations on unprotected nucleosides where theunprotected nucleosides are highly insoluble in non-polar organicsolvents.

SUMMARY OF THE INVENTION

The present invention overcomes the difficulties and shortcomings of theprior art with regard to the synthesis 2-halo-6-aminopurine compoundsand derivatives thereof and especially of 2-halogenated purineribonucleosides and 2-halogenated-2′- and 3′-deoxy and 2′ and3′-substituted purine ribonucleosides. Disclosed herein are methods forproducing 2-halo-6-aminopurine compounds and derivatives thereof andespecially 2-halogenated-2′-deoxy purine nucleosides, 2-halogenatedpurine ribonucleosides, and 2′ and 3′-substituted analogs thereof viahalogenation at the 2 position in a unique organic solvent system atroom temperature.

Thus, in one aspect the invention relates to methods for producing2-halo-6-amino derivatives, comprising the steps of:

admixing a nonpolar aprotic organic solvent with a polar aprotic organicsolvent to produce a solvent mixture;

dissolving in the solvent mixture a compound having the formula

where R is selected from the group consisting of hydrogen, C₁ to C₂₀alkyl, including linear and branched chain alkyl, cycloalkyl,alkoxyalkyl, alkylamino, ether, thioether, haloalkyl, a monocyclic arylgroup, a multicyclic aryl group, a heterocyclic aryl group having from 1to 20 carbon atoms and 1 to 10 heteroatoms, sugar moieties selected fromthe group consisting of β-D-ribofuranosyl, deoxy-β-D-furanosyl,xylofuranosyl, arabinofuranosyl, and 2′-, 3′-, and 2′,3′-substituted orderivatized analogs of β-D-ribofuranosyl, deoxy-β-D-furanosyl,xylofaranosyl, and arabinofuranosyl sugar moieties; and

reacting the compound in the solvent mixture with an organic nitrite anda metal halide, where the metal halide is a Lewis acid, to produce areaction product.

In another aspect the invention relates to the methods for producing2-halonucleosides comprising the steps of:

admixing a nonpolar aprotic organic solvent with a polar aprotic organicsolvent to produce a solvent mixture;

dissolving in the solvent mixture a nucleoside having the formula

 where Q is O or S;

where R¹ and R² together form a moiety with the formula O—A(Y)—O, whereA is C, S, or P—R and where Y is O, S, N—R, or 2R;

or where R¹ and R² are independently hydrogen, O—R, R, N—R₂, N₃, X, orS—R;

where R is hydrogen, C₁ to C₂₀ alkyl, including linear and branchedchain alkyl, cycloalkyl, alkoxyalkyl, alkylamino, ether, thioether,haloalkyl, a monocyclic aryl group, a multicyclic aryl group, or aheterocyclic aryl group having from 1 to 20 carbon atoms and 1 to 10heteroatoms and where X is Cl, Br, F, or I; and

reacting the nucleoside in the solvent mixture with an organic nitriteand a metal halide, where the metal halide is a Lewis acid, to produce areaction product.

In yet another aspect the invention relates to the methods for producing2-halo-6-aminoalkyloxy derivatives comprising the steps of:

admixing a nonpolar aprotic organic solvent with a polar aprotic organicsolvent to produce a solvent mixture;

dissolving in the solvent mixture a nucleoside analog having the formula

 where R¹ is hydrogen, CH₂OH, or CH₂OPOM; R² is OH, OPh, or OPOM; andR³is OH, OPh, or OPOM;

or where R¹ and R² form the moiety —CH₂O— and R³ is OH, wherein POM ispivalyloxymethyl; and

reacting the nucleoside analog in the solvent mixture with an organicnitrite and a metal halide, where the metal halide is a Lewis acid, toproduce a reaction product.

In one particular aspect, the invention relates to methods for producing2-halo-6-aminopurine-2′-deoxy or 2′-substituted nucleosides comprisingthe steps of:

admixing a nonpolar aprotic organic solvent with a polar aprotic organicsolvent to produce a solvent mixture;

dissolving in the solvent mixture an unprotected nucleoside having theformula

 where R¹ is hydrogen, OR, R, NR₂, N₃, X, or SR; R is hydrogen, C₁ toC₂₀ alkyl, including linear and branched chain alkyl, cycloalkyl,alkoxyalkyl, alkylamino, ether, thioether, haloalkyl, a monocyclic arylgroup, a multicyclic aryl group, or a heterocyclic aryl group havingfrom 1 to 20 carbon atoms and 1 to 10 heteroatoms, and X is Cl, Br, F,or I; and

reacting the unprotected nucleoside in the solvent mixture with anorganic nitrite and a metal halide, where the metal halide is a Lewisacid, to produce a reaction product.

In another aspect, the invention relates to methods for producing2-halo-6-aminopurine-3′-deoxy or 3′-substituted nucleosides comprisingthe steps of: admixing a nonpolar aprotic organic solvent with a polaraprotic organic solvent to produce a solvent mixture;

dissolving in the solvent mixture an unprotected nucleoside having theformula

 where R¹ is hydrogen, OR, R, NR₂, N₃, X, or SR; R is hydrogen, C₁ toC₂₀ alkyl, including linear and branched chain alkyl, cycloalkyl,alkoxyalkyl, alkylamino, ether, thioether, haloalkyl, a monocyclic arylgroup, a multicyclic aryl group, or a heterocyclic aryl group havingfrom 1 to 20 carbon atoms and 1 to 10 heteroatoms, and X is Cl, Br, F,or I; and

reacting the unprotected nucleoside in the solvent mixture with anorganic nitrite and a metal halide, wherein the metal halide is a Lewisacid, to produce a 2-halo-6-aminopurine reaction product.

In a further aspect, the present method comprises a method forstabilizing the 2-haloadenosine and 2-halo-deoxyadenosine reactionproducts produced by the synthetic methods of the present inventioncomprising subjecting the reaction products to resin columnchromatography.

In another aspect, the invention relates to methods for producing2-halo-6-aminopurine-2′,3′-dideoxy nucleosides comprising the steps of:admixing a nonpolar aprotic organic solvent with a polar aprotic organicsolvent to produce a solvent mixture;

dissolving in the solvent mixture an unprotected nucleoside having theformula

 where Q is O or S; and

reacting the unprotected nucleoside in the solvent mixture with anorganic nitrite and a metal halide, wherein the metal halide is a Lewisacid, to produce a 2-halo-6-aminopurine reaction product.

Another aspect the invention relates to the methods for producing2-halo-6-aminopurine-4-thionucleosides comprising the steps of:

admixing a nonpolar aprotic organic solvent with a polar aprotic organicsolvent to produce a solvent mixture;

dissolving in the solvent mixture a 4-thionucleoside having the formula

 where R¹ and R² are independently hydrogen, OR, R, NR₂, N₃, X, or SR;

where R is linear or branched chain alkyl, cycloalkyl, alkoxyalkyl,ether, thioether, haloalkyl, a monocyclic aryl group, a multicyclic arylgroup, or a heterocyclic aryl group having from 1 to 20 carbon atoms and1 to 10 heteroatoms, and X is Cl, Br, F, or I; and

reacting the nucleoside in the solvent mixture with an organic nitriteand a metal halide, where the metal halide is a Lewis acid, to produce areaction product.

Yet another aspect the invention relates to the methods for producing2-halo-6-aminopurine-2′,3′-derivatized nucleosides comprising the stepsof:

admixing a nonpolar aprotic organic solvent with a polar aprotic organicsolvent to produce a solvent mixture;

dissolving in the solvent mixture a nucleoside having the formula

 where Q is O or S;

where R¹ and R² together form a moiety with the formula O—A(Y)—O, whereA is C, S, or P—R and where Y is O, S, N—R, or 2R;

or where R¹ and R² are independently hydrogen, O—R, R, N—R₂, N₃, X, orS—R; where R is linear or branched chain alkyl cycloalkyl, alkoxyalkyl,ether, thioether, haloalkyl, a monocyclic aryl group, a multicyclic arylgroup, or a heterocyclic aryl group having from 1 to 20 carbon atoms and1 to 10 heteroatoms, and X is Cl, Br, F, or I;

reacting the nucleoside in the solvent mixture with an organic nitriteand a metal halide, where the metal halide is a Lewis acid, to produce areaction product.

In one particular aspect, the invention relates to methods for producing2-halo-6-aminopurine-2′-deoxy or 2′-substituted N-7 glycosylatednucleosides comprising the steps of:

admixing a nonpolar aprotic organic solvent with a polar aprotic organicsolvent to produce a solvent mixture;

dissolving in the solvent mixture an unprotected N-7 glycosylatednucleoside having the formula

 where Q is O or S;

where R¹ and R² together form a moiety with the formula O—A(Y)—O, whereA is C, S, or P—R and where Y is O, S, N—R, or 2R;

or where R¹ and R² are independently hydrogen, O—R, R, N—R₂, N₃, X, orS—R;

where R is linear or branched chain alkyl, cycloalkyl, alkoxyalkyl,ether, thioether, haloalkyl, a monocyclic aryl group, a multicyclic arylgroup, or a heterocyclic aryl group having from 1 to 20 carbon atoms and1 to 10 heteroatoms, and X is Cl, Br, F, or I; and

reacting the unprotected nucleoside in the solvent mixture with anorganic nitrite and a metal halide, where the metal halide is a Lewisacid, to produce a reaction product.

In yet another aspect, the present invention is directed to novelmorpholino 2-halopurines of the formula:

where X is fluorine, chlorine, bromine, or iodine and R¹ is alkyl, aryl,substituted aryl, aryloxy, or substituted aryloxy, and methods forsynthesizing such compounds.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and literature references cited hereinare incorporated by reference in their entirety. In case of a conflictin terminology, the present specification controls.

Definitions

The following terms generally have the following meanings.

The term “aryl” refers to aromatic groups, which have at least one ringhaving a conjugated pi electron system, including for examplecarbocyclic aryl, heterocyclic aryl and biaryl groups, all of which maybe optionally substituted. Carbocyclic aryl groups are groups whereinall the ring atoms on the aromatic ring are carbon atoms, such asphenyl. Also included are optionally substituted phenyl groups, beingpreferably phenyl or phenyl substituted by one to three substituents.Further included are phenyl rings fused with a five or six memberedheterocyclic aryl or carbocyclic ring, optionally containing one or moreheteroatoms such as oxygen, sulfur, or nitrogen. Where chemical groupsor moieties are indicated to be “optionally substituted”, it is meantthat the groups can be chemically bonded to one or more other chemicalgroups, such groups preferably being, but not limited to, lower alkyl,hydroxy, lower alkoxy, lower alkanoyloxy, halogen, cyano, perhalo loweralkyl, lower acylamino, lower alkoxycarbonyl, amino, alkylamino,carboxamido, and sulfamido.

Heterocyclic aryl groups are monocyclic or polycyclic groups having from1 to 10 heteroatoms as ring atoms in the aromatic rings and theremainder of the ring atoms carbon atoms. Suitable heteroatoms includeoxygen, sulfur, and nitrogen. Heterocyclic aryl groups include furanyl,thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, and thelike, all optionally substituted.

Optionally substituted furanyl represents 2- or 3-furanyl or 2- or3-furanyl preferably substituted by lower alkyl or halogen. Optionallysubstituted pyridyl represents 2-, 3- or 4-pyridyl or 2-, 3- or4-pyridyl preferably substituted by lower alkyl or halogen. Optionallysubstituted thienyl represents 2- or 3-thienyl, or 2- or 3-thienylpreferably substituted by lower alkyl or halogen.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,and may be optionally substituted.

The term “lower” referred to herein in connection with organic radicalsor compounds respectively defines such with up to and including 7,preferably up to and including 4 and advantageously one or two carbonatoms. Such groups may be straight chain or branched.

The terms (a) “alkylamino”, (b) “arylamino”, and (c) “aralkylamino”,respectively, refer to the groups —NRR′ wherein respectively, (a) R isalkyl and R′ is hydrogen, aryl or alkyl; (b) R is aryl and R′ ishydrogen or aryl, and (c) R is aralkyl and R′ is hydrogen or aralkyl.

The term “acylamino” refers to RC(O)NR′.

The term “carbonyl” refers to —C(O)—.

The term “acyl” refers to RC(O)— where R is alkyl, aryl, aralkyl, oralkenyl.

The term “carboxamide” or “carboxamido” refers to —CONRR wherein each Ris independently hydrogen, lower alkyl or lower aryl.

The term “alkyl” refers to saturated aliphatic groups includingstraight-chain, branched chain and cyclic groups, optionally containingone or more heteroatoms.

The invention in one aspect relates to a novel process for preparing thecompound 2-CdA and other 2-halogenated purine nucleosides and2-halogenated 2′-deoxy and 2′ substituted nucleosides having theformula:

where R¹ is hydrogen, OR, R, NR₂, N₃, X, or SR; R is hydrogen, C₁ to C₂₀alkyl, including linear and branched chain alkyl and cycloalkyl,alkoxyalkyl, alkylamino, ether, thioether, haloalkyl, a monocyclic arylgroup, a multicyclic aryl group, or a heterocyclic aryl group havingfrom 1 to 20 carbon atoms and 1 to 10 heteroatoms, and X is Cl, Br, F,or I.

The method comprises admixing a nonpolar aprotic organic solvent with apolar aprotic organic solvent to produce a solvent mixture, dissolvingor suspending in the solvent mixture an unprotected nucleoside havingthe formula:

where R¹ is hydrogen, OR, R, NR₂, N₃, X, or SR; R is hydrogen, C₁ to C₂₀alkyl, including linear and branched chain alkyl, cycloalkyl,alkoxyalkyl, alkylamino, ether, thioether, haloalkyl, a monocyclic arylgroup, a multicyclic aryl group, or a heterocyclic aryl group havingfrom 1 to 20 carbon atoms and 1 to 10 heteroatoms, and X is Cl, Br, F,or I; and reacting the unprotected nucleoside in the solvent mixturewith an organic nitrite and a metal halide, where the metal halide is aLewis acid, to produce a reaction product.

Methods for the synthesis of the 2′-deoxy, 2′-OH, and 2′-O-alkylnucleoside diaminopurine starting materials are known in the art.Syntheses of many exemplary starting materials are described in suchworks as: Chemistry of Nucleosides and Nucleotides, Vol. 1, Ed.Townsend, L. B., Plenum Press, New York, N.Y., 1988; Chemistry ofNucleosides and Nucleotides, Vol 2., Ed. Townsend, L. B., Plenum Press,New York, N.Y., 1991; Chemistry of Nucleosides and Nucleotides, Vol. 3,Ed. Townsend, L. B., Plenum Press, New York, N.Y., 1994; OligonucleotideSynthesis: A Practical Approach, Ed. Gait, M. J., Oxford Univ. Press,New York, N.Y., 1984; Oligonucleotides and Analogues: A PracticalApproach, Ed. Eckstein, F., Oxford Univ. Press, New York, N.Y., 1991.Syntheses of such starting materials are also described, e.g., in U.S.Pat. Nos. 5,506,351 and 5,571,902.

Methods for the synthesis of 2′-fluoro and other 2′-haloribonucleosides(i.e., where R¹ is halogen) are described in, e.g., U.S. Pat. No.5,420,115, European Patent application 417999; Tuttle, J. V.; Tisdale,S. M. and Krenitsky, T. A., J. Med. Chem., 36(1): 119-125, 1993; Thomas,H. J.; Tiwari, K. N.; Clayton, S. J.; Secrist, J. A. III and Montgomery,J. A., Nucleosides and Nucleotides, 13 (1-3): 309-323, 1994. Startingmaterials for the synthesis of compounds of the invention where R¹ is N₃can be obtained by substitution of 2′-halo nucleosides. Startingmaterials for the synthesis of compounds of the invention where R¹ isNH—R can be obtained by reduction of these N₃ compounds.

In a preferred embodiment, X is bromine, chlorine, fluorine, or iodine,and R¹ is hydrogen.

In other preferred embodiments, X is bromine, chlorine, fluorine, oriodine, and R¹ is hydroxyl or is OR, where R is hydrogen, C₁ to C₂₀alkyl, including linear and branched chain alkyl, cycloalkyl,alkoxyalkyl, alkylamino, ether, thioether, haloalkyl, a monocyclic arylgroup, a multicyclic aryl group, or a heterocyclic aryl group havingfrom 1 to 20 carbon atoms and 1 to 10 heteroatoms.

In a most preferred embodiment, X is chlorine and R¹ is hydrogen.

In another aspect, the invention is directed to methods for synthesizing2-halogenated purine 3′-O-alkyl, 3′-deoxy, and 3′-substitutedribonucleosides. The methods comprise admixing a nonpolar aproticorganic solvent with a polar aprotic organic solvent to produce asolvent mixture, dissolving or suspending in the solvent mixture anunprotected nucleoside having the formula:

where R¹ is hydrogen, OR, R, NR₂, N₃, X, or SR; R is hydrogen, C₁ to C₂₀alkyl, including linear and branched chain alkyl, cycloalkyl,alkoxyalkyl, alkylamino, ether, thioether, haloalkyl, a monocyclic arylgroup, a multicyclic aryl group, or a heterocyclic aryl group havingfrom 1 to 20 carbon atoms and 1 to 10 heteroatoms, and X is Cl, Br, F,or I; and reacting the unprotected nucleoside in the solvent mixturewith an organic nitrite and a metal halide, where the metal halide is aLewis acid, to produce a reaction product.

Methods for the synthesis of 3′-O-alkyl starting materials are known andwidely reported in the art and are described, e.g., in U.S. Pat. No.5,506,351. The synthesis of unprotected 3′-deoxyribonucleoside is setout in Kumar, A.; Khan, S. I.; Manglani, A.; Khan, Z. K. and Katti, S.B., Nucleosides & Nucleotides 13(5): 1049-1058, 1994. Methods for thesynthesis of 3′-substituted nucleosides, e.g.,3′-Fluoro-3′-deoxyribonucleoside are described in Koshida, R.; Cox, S.;Harmenberg, J.; Gilljam, G. and Wahren, B., Antimicrob. AgentsChemother., 33(12): 2083-2088, 1989. Exemplary methods for the synthesisof other 3′-substituted nucleosides, such as3′-Amino-3′-deoxyribonucleoside are set forth in Kissman, H. M.;Hoffman, A. S.; Weiss, M. J. J. Med. Chem. 6(4): 407-409, 1963; Goldman,L.; Marsico, J. W.; Weiss, M. J. J. Med. Chem. 6(4): 410-412, 1963;Goldman, L.; Marsico, J. W.; J. Med. Chem. 6(4), 413-423, 1963; Soenens,J.; Francois, G.; Van den Eeckhout, E.; Herdewijn, P., Nucleosides &Nucleotides 14(3-5): 409-411, 1995; and Pannecouque, C.; Van Poppel, K.;Balzarini, J.; Claes, P.; De Clercq, E.; Herdewijn, P., Nucleosides &Nucleotides 14(3-5): 541-544, 1995).

Methods for the halogenation of 2,6 diaminonucleosides coupled to sugarmoieties such as arabinose and xylose are also within the scope of thepresent invention. Exemplary syntheses of such starting materials can befound, e.g., in Montgomery, J. A. and Hewson, K., J. Med. Chem., 12:498-504, 1969; Hansske, F.; Madej, D. and Robins, M. J., Tetrahedron,40(1): 125-135, 1984 (xylose and arabinose); Krenitsky, T. A., Koszalka,G. W.; Tisdale, J. V.; Rideout, J. L. and Elion, G. B., Carbohydr. Res.,97(1): 139-146, 1981; Krenitsky, T. A., Elion, G. B. and Rideout, J. E.,EP 790613; Utagawa, T.; Miyoshi, T.; Morisawa, H.; Yamazaki, A.;Yoshinaga, F. and Mitsugi, K., DE 2835151; Elion, G. B. and Strelitz, R.A., U.S. Pat. No. 4,038,479; Wellcome Foundation, GB 1,386,584; Elion,G. B.; Litster, J. E. and Beachamp, L. M. III, DE 2156637; Elion, G. B.and Strelitz, R. A., DE 205637. Exemplary syntheses of 2′-substitutedarabinonucleoside starting materials can be found in, e.g., Robins, M.J.; Zou, R.; Hansske, F. and Wnuk, S. F., Can. J. Chem. 75(6): 762-767,1997; Watanabe, K. A.; Pankiewicz, K. W.; Krzeminski, J. and Nawrot, B.,WO 9211276A1; Tuttle, J. V. and Krenitsky, T. A., EP 285432A2; Watanabe,K. A.; Chu, C. K. and Fox, J. J., EP 219829A2; and Montgomery, J. A.;Shortnacy, A. T.; Carson, D. A. and Secrist, J. A. III, J. Med. Chem.,29(11): 2389-2392, 1986.

Methods for synthesizing 2-halogenated purine 2′,3′-dideoxy nucleosidescomprise admixing a nonpolar aprotic organic solvent with a polaraprotic organic solvent to produce a solvent mixture, dissolving orsuspending in the solvent mixture an unprotected nucleoside having theformula

and reacting the unprotected nucleoside in the solvent mixture with anorganic nitrite and a metal halide, where the metal halide is a Lewisacid, to produce a reaction product.

Such starting compounds can be prepared according to well-known methodsof dideoxy nucleoside syntheses such as those disclosed by Webb II, R.R.; Wos, J. A.; Martin, J. C.; Brodfuehrer, P. R. Nucleosides &Nucleotides, 7(2): 147-153, 1988; Prisbe, E. J.; Martin, J. C. Synth.Comm. 15(5): 401-409 1985; Horwitz, J. P.; Chua, J.; Da Rooge, M. A.;Noel, M.; Klundt, I. L. J. Org. Chem. 31: 205-211, 1966; Horwitz, J. P.;Chua, J.; Noel, M.; Donatti, J. T. J. Org. Chem. 32: 817-818, 1967.

Methods for synthesizing 2-halo 6-aminopurine-4′-thionucleosidescomprise admixing a nonpolar aprotic organic solvent with a polaraprotic organic solvent to produce a solvent mixture, dissolving orsuspending in the solvent mixture an unprotected nucleoside having theformula

where R¹ and R² are independently hydrogen, OR, R, NR₂, N₃, X, or SR;where R is hydrogen, C₁ to C₂₀ alkyl, including linear and branchedchain alkyl, cycloalkyl, alkoxyalkyl, alkylamino, ether, thioether,haloalkyl, a monocyclic aryl group, a multicyclic aryl group, or aheterocyclic aryl group having from 1 to 20 carbon atoms and 1 to 10heteroatoms, and X is Cl, Br, F, or I; and reacting the unprotectednucleoside in the solvent mixture with an organic nitrite and a metalhalide, where the metal halide is a Lewis acid, to produce a reactionproduct.

Such starting materials can be prepared according to well-known methodsfor 4-thionucleosides synthesis, such as those disclosed in Leydier, C.;Bellon, L.; Barascut, J. -L.; Imbach, J. -L. Nucleosides & Nucleotides,14(3-5): 1027-1030, 1995; Bellon, L.; Leydier, C.; Barascut, J. -L.;Imbach, J. -L. Nucleosides & Nucleotides 12(8): 847-852, 1993, Bellon,L.; Barascut, J. -L.; Imbach, J. -L. Nucleosides & Nucleotides 11(8):1467-1479, 1992; and Reist, E. J.; Gueffroy, D. E.; Goodman, L. Chem.Ind. (London), 1364, 1964.

Methods for synthesizing 2-halo 6-aminopurine-2′,3′-derivatizedribonucleosides comprise admixing a nonpolar aprotic organic solventwith a polar aprotic organic solvent to produce a solvent mixture,dissolving or suspending in the solvent mixture an unprotectednucleoside having the formula

where Q is O or S; where R¹ and R² together form a moiety with theformula O—A(Y)—O, where A is C, S, or P—R and where Y is O, S, N—R, or2R; where R is hydrogen, C₁ to C₂₀ alkyl, including linear and branchedchain alkyl, cycloalkyl, alkoxyalkyl, alkylamino, ether, thioether,haloalkyl, a monocyclic aryl group, a multicyclic aryl group, or aheterocyclic aryl group having from 1 to 20 carbon atoms and 1 to 10heteroatoms, and X is Cl, Br, F, or I; and reacting the unprotectednucleoside in the solvent mixture with an organic nitrite and a metalhalide, where the metal halide is a Lewis acid, to produce a reactionproduct.

Starting compounds for such methods, such as isopropylidene nucleosides,can be prepared based upon the methods described in, e.g., Schmidt, O.Th. Methods Carbohydr. Chem., II, 318 (1963); de Belder, A. N. Adv.Carbohydr. Chem. 20: 219, (1965); Hampton, A. J. Amer. Chem. Soc., 83:3640, 1961; Davis, J. T.; Tirumala, S.; Jenssen, J. R.; Radler, E.;Fabris, D. J. Org. Chem., 60: 4167, 1995; Chladek, S.; Smrt, J. Collect.Czech. Chem. Commun. 28: 1301-1308, 1963; and Anzai, K.; Matsui, M.Bull. Chem. Soc. Jpn. 47: 417-420, 1974. Exemplary syntheses of2′3′thionocarbonate nucleosides are described, e.g., in Anzai, K.;Matsui, M. Agric. Biol. Chem. 37: 345-348, 1973.

Methods for halogenation of 2,6 diaminonucleosides coupled to sugarderivatives at the N-7 position of the base comprise admixing a nonpolaraprotic organic solvent with a polar aprotic organic solvent to producea solvent mixture, dissolving or suspending in the solvent mixture anunprotected nucleoside having the formula.

where R¹ and R² are independently hydrogen, OR, R, NR₂, N₃, X, SR; whereQ is O or S; where R is hydrogen, C₁ to C₂₀ alkyl, including linear andbranched chain alkyl, cycloalkyl, alkoxyalkyl, alkylamino, ether,thioether, haloalkyl, a monocyclic aryl group, a multicyclic aryl group,or a heterocyclic aryl group having from 1 to 20 carbon atoms and 1 to10 heteroatoms, and X is Cl, Br, F, or I; and reacting the unprotectednucleoside in the solvent mixture with an organic nitrite and a metalhalide, where the metal halide is a Lewis acid, to produce a reactionproduct.

Starting materials having the formula above are known, and can beprepared, e.g., according to the methods set out in Worthington, V. L.,Fraser, W., and Schwalbe, C. H.; Carbohydrate Research, 275: 275-284,1995. N-7 to N-9 or N-9 to N-7 glycosyl transfer reactions are describedin the art, e.g., see Seela, F.; Winter, H. Nucleosides & Nucleotides,14(1&2): 129-142, 1995.

The synthesis of 2-halo-6-aminopurines that have acyclic moieties linkedat the N-9 position of the base comprise admixing a nonpolar aproticorganic solvent with a polar aprotic organic solvent to produce asolvent mixture, dissolving or suspending in the solvent mixture anunprotected diaminopurine that has an acyclic moiety linked at the N-9position of the base, and reacting the unprotected compound in thesolvent mixture with an organic nitrite and a metal halide, where themetal halide is a Lewis acid, to produce a reaction product.

Synthesis of diaminopurines that have an acyclic moiety linked at theN-9 position are well-known by those of ordinary skill in the art, andare described, e.g., by Holy, A. and Dvorakova, H., Nucleosides &Nucleotides 14(3-5): 695-702, 1995; Holy, A.; Dvorakova, H andMasojidkova, M., Collect. Czech. Chem. Commun., 60(8): 1390-1409, 1995;Holy, A.; Dvorakova, H.; de Clercq, E.; Desire, A. and Balzarini, J. M.H., WO 9403467; Rosenberg, I. and Holy, A.; Dvorakova, H., Collect.Czech. Chem. Commun., 54(8): 2190-2210, 1989; de Clercq, E.; Holy, A.and Rosenberg, I., Antimicrob. Agents Chemother., 33(2): 185-191, 1989;Yokota, T.; Mochizuki, S.; Konno, K.; Mori, S.; Shigeta, S. and deClercq, E., Nucleic Acids Symposium Series 22, 17-18, 1990; Holy, A.;Rosenberg, I.; Dvorakova, H. and de Clercq, E., Nucleosides &Nucleotides 7(5-6): 667-670, 1988; Holy, A., Collect. Czech. Chem.Commun., 58(3): 649-674, 1993; Holy, A.; Rosenberg, I. and Dvorakova,H., Collect. Czech. Chem. Commun., 54(9): 2470-2501, 1989; Holy, A. andRosenberg, I., CS 263955; Holy, A.; Rosenberg, I. and de Clercq, E., EP253412; and Alexander, P. and Holy, A., Collect. Czech. Chem. Commun.,58(5): 1151-1163, 1993.

Scheme 1, below, sets forth an exemplary process of the presentinvention, the preparation of 2-CdA. The process utilizes 2,6-diaminopurine deoxyriboside (2-amino deoxyadenosine or “DAPD”) as a startingmaterial. DAPD can be prepared by methods reported in U.S. Pat. No.5,506,351 and in Seela and Gabler, Helv. Chim. Acta., 77: 622, 1994.DAPD is also commercially available from Reliable BiopharmaceuticalCorporation, St. Louis, Mo.

DAPD is suspended in a novel solvent combination which includes a polaraprotic organic solvent and a nonpolar aprotic organic solvent,(DMSO/dichloroethane is shown in Scheme 1), in a ratio of from about1:10 to 10:1, and is cooled to 0° C. under an inert atmosphere, e.g.,nitrogen or argon. Following the cooling step, the DAPD is diazotized atthe 2-position with an organic nitrite, e.g., tert-butyl nitrite, andhalogenated with a metal halide Lewis acid, e.g., antimony trichloride,at room temperature. The reaction produces 2-CdA in high yield. Thereaction is unlike any other reactions for diazotization/substitution atthe 2 position of a 2-amino purine nucleoside or 2-amino-2′-deoxy purinenucleoside because the diazotization is performed on a unprotectednucleoside. It is the selective combination of solvents in a particularratio that allows the diazotization to proceed; the diazotization is akey step in this transformation. If only a polar aprotic organic solventis used in the method, the diazotization reaction does not occur.Similarly, if only a nonpolar aprotic organic solvent is used in themethod, the diazotization reaction fails. The chloride transfer, whichemploys metal halide Lewis acids, such as SbCl₃, is very efficient.

As indicated above, the role of the solvent in the halogenation reactionhas been found to be critical. A series of TBN/SbCl₃ chlorinationreactions using a number of aprotic polar organic solvents and non-polarorganic solvents with varying solvent ratios were performed. It wasdetermined that the reaction proceeds when the ratio of aprotic polarorganic solvent to non-polar organic solvent is between 10:1 and 1:10.The preferred solvent ratio is 1 part aprotic polar organic solvent to 4parts non-polar organic solvent. The reaction will not proceed when 100%non-polar solvent is used, nor will the reaction proceed when 100%aprotic polar organic solvent is used. Thus, the present inventors havediscovered that there is a specific combination of solvents in aparticular solvent component ratio range that is required for asuccessful halogenation reactions.

After the diazotization/substitution reaction is complete the reactionmixture is typically dried, using, e.g., a rotary evaporator, andneutralized. Subsequently, the crude product should immediately bestabilized by chromatography over a DVB (divinylbenzene)-cross-linkedpolystyrenic resin column, e.g., Amberchrom CG161 (Rohm & Haas,Philadelphia, Pa.). If this product-stabilizing column chromatographystep is not performed, and the crude reaction mixture is stored, even at0° C., the product will degrade over a period of about two days. Thestabilized product can be further purified by subjecting it to a strongcation exchange resin column chromatography step, e.g., Dowex,(available from many commercial suppliers, such as Sigma Chemical, St.Louis, Mo. or Supelco, Bellefonte, Pa.) and the pure product, e.g.,2-CdA, can then be recrystallized from water.

The Amberchrom CG161 XUS resin column is prepared, typically so thatabout 4 g of 2-CldA per L of resin is applied. Two-thirds of the resinis removed from the column and slurried with the reaction mixture andthen loaded onto the column. The column is washed with water at a flowrate of about 30 to about 80 mL/min until the absorbance at 265 nm dropsbelow about 5 or until the 2-CldA begins to elute off the column andthen 3 to 6 L fractions are collected. This is followed with elutionwith 5% methanol in water, and then 30% methanol in water until theabsorbance of the eluate at 265 nm is below 10. All fractions containinggreater than 75% 2Cl-dA by peak area using HPLC are collected andconcentrated on a rotary evaporator at 45° C. The collected fractionsare concentrated to a gum, which is at a stable state and can be leftfor up to 7 days if necessary.

The gum is dissolved in water and then concentrated to a concentrationof 12-15 g of 2-CldA/L. This solution is then charged onto a Dowex 1×4anion exchange resin column. The column is eluted at about 140-240mL/min, first with water until the absorbance of the eluate at 265 nm isbelow 10, followed by 10% methanol in water, 15% methanol in water, andfinally a 25% methanol in water wash until all of the 2-CldA has elutedoff the column. Fractions of about 2-6 L each containing 2-CldA arecollected and assayed for purity by HPLC. The fractions thatcontain >90% pure 2Cl-dA (by HPLC) is adjusted to pH 7.5 or greater byusing 1.0M sodium bicarbonate solution to achieve a concentration 10 mMsodium bicarbonate. These are saved individually, without pooling. Asecond Dowex 1×4 anion exchange resin column is prepared, with about 1 Lof resin per 10 g of 2-CldA. The column is equilibrated in 10 mM sodiumbicarbonate solution before the 2-CldA charge is loaded. The charge ismade up of the fractions from the previous 1×4 column. The fractions areloaded onto the 1×4 column in the order that they eluted off the first1×4 column. After the column is loaded, the first eluant, 10 mM sodiumbicarbonate in water, is started over the column. Once the majority ofthe 2-CldA is eluted off the column, the buffer is switched to 10%methanol in a 10 mM sodium bicarbonate solution. The column is washedwith this eluant until 90% or more of 2-CldA has eluted off the column.All fractions that contain 2-CldA at greater than 98% purity by HPLC areconcentrated in a rotary evaporator at 45° C. under vacuum.

The main (>98% pure) product pool is evaporated to a concentration ofabout 22 g 2-CldA per liter. The concentrated pool is filtered using aBuchner funnel and Whatman 54 hardened filter paper. The filteredsolution is sealed and placed in a refrigerator for 17 hours at about 8to 14° C. for crystallization. The concentrated solution should remainat about 8 to 14° C. for at least 14 hours. The crystallized 2-CldA iscollected by filtration. The solids are washed with about 50 to 150 mLof cold water, i.e, at from about 5 to 10° C. Both the solids and thefiltrate are assayed for 2-CldA using HPLC.

The crystallized solids are redissolved in 10 mM sodium bicarbonatesolution at approximately 4 to 5 grams of 2-CldA per liter and thenconcentrated down to about 22 grams per liter using the rotaryevaporator. The concentrated solution is then filtered and placed in arefrigerator for about 17 hours, or overnight for recrystallization.After about 17 hours, or overnight, the solids are filtered and both thesolids and filtrate are assayed by HPLC.

If there is a significant amount of 2-CldA in the filtrate from thefinal recrystallization which is greater than 99% purity by HPLC, thenthe filtrate is concentrated and recrystallized to recover 2-CldA. Allthe product is 99% or greater pure is placed in a vacuum oven at atemperature of between about 37° and 45° C. for about 24 hours to dry.After drying the crystals are ground up and dried until the watercontent is about 2% or less by weight. The crystals are weighed for afinal yield.

For maximum recovery, fractions with <98% pure 2-CldA from the secondDowex column are pooled and reprocessed over another 1×4 Dowex column.These clean [>98%] fractions are combined with the filtrates fromprevious recrystallizations. This solution is concentrated andrecrystallized as described.

The product can be analyzed by any of the common structural analysismethods familiar to those of skill in the art, such as nuclear magneticresonance (NMR), ultraviolet (UV), or infrared (IR) spectroscopy, or byelemental analysis and optical rotation. The overall yield of 2-CdA fromDAPD achievable by the methods of the present invention is approximatelyten fold (1000%) greater than obtained by the methods reported in theprior art, e.g., U.S. Pat. No. 5,208,327. Thus, the methods of thepresent invention are clearly superior in yield and simplicity relativeto the methods disclosed by the prior art.

The improved methods of the present invention also permit the synthesisof novel 2-halo-6-aminopurine morpholino compounds. These morpholinocompounds are useful in, e.g., synthesizing polymers which can bindspecifically to polynucleotides with specific sequences. Thus, they areuseful for the detection of specific sequences of polynucleotides, andare potentially useful as inactivators of specific genetic sequences.

The novel 2-halo-6-aminopurine morpholino compounds of the inventionhave the formula:

where X is F, Cl, Br, or I and where R¹ is aryl, alkyl, aklyoxy oraryloxy. The novel morpholino compounds of the invention can beprepared, e.g., according to Scheme 2, below.

A 2-amino-6-halopurine nucleoside, prepared according to the methodsdisclosed above and in the Examples, below, is first N-protected with acarboxylic acid chloride and this intermediate is treated with sodiumperiodate, which cleaves the ribose sugar ring between the 2′ and 3′carbons, yielding a dialdehyde. The dialdehyde is reacted with ammonia,which results in a morpholino ring having 2′ and 3′ hydroxyl groups.Sodium cyanoborohydride treatment reduces the ring hydroxyl groups,yielding the novel morpholino compounds of the invention. The synthesisof other morpholinopurines are disclosed, e.g., in U.S. Pat. Nos.5,185,444 and 5,521,063.

The following non-limiting examples are provided to illustrate theinvention. Modifications and variations of the methods and compoundsdisclosed herein will be apparent to those of ordinary skill in the art,and are intended to be within the scope of the invention.

EXAMPLE 1 Synthesis of 2-chloro-2′deoxyadenosine

To a three-neck 12 L reaction flask equipped with a stir bar,temperature probe, and drying tube was added dry diaminopurinedeoxyriboside (DAPD, 60 g, 0.225 mol) and 900 ml DMSO. The mixture wasstirred until the DAPD dissolved completely and then dichloroethane(4200 ml) was added. The solution was cooled to below 10° C. using anice bath and then antimony trichloride (24 g, 0.105 mol) was added. Tothe reaction mixture was then added tert-butyl nitrite (55.4 mL, 48 g,0.466 mol) dropwise over 10 minutes. After 20 minutes stirring, a secondaliquot of antimony trichloride (24 g, 0.105 mol) was added, followed bya final third aliquot of antimony trichloride (24 g, 0.105 mol) after anadditional 20 minutes. The reaction was stirred overnight at roomtemperature. When less than 5% diaminopurine deoxyriboside (by peak areausing HPLC [Novapak C-18; 5% acetonitrile/water, 0.1M TEA, pH 7 withacetic acid]) remained, the reaction was quenched with ˜102 mLtriethylamine in approximately 35 mL aliquots until the pH was 7. Thereaction mixture was evaporated at 40° C. under reduced pressure todryness, placed in an ice bath, stirred vigorously and treated slowlywith 3200 mL of 0.1M sodium bicarbonate solution.

EXAMPLE 2 Stabilization and isolation of 2-chloro-2′-deoxyadenosine

The neutralized reaction mixture was immediately applied to apolystyrene-divinylbenzene crosslinked polymeric resin column(Amberchrom CG161 md, 2500 g) to prevent product degradation. The columnwas eluted with water, 5% MeOH/water and 30% MeOH/water and thecollected fractions were analyzed. Fractions containing >75% 2-CdA werepooled and concentrated to dryness. The crude product residue from theAmberchrom column was dissolved in water and loaded on a polystyreneanion exchange resin (Dowex 1×4 200-400 mesh, 500 ml) column. The columnwas eluted with water, 10% MeOH/water, 15% MeOH/water and 25%MeOH/water, and the eluate was collected from the column in fractionswas analyzed by HPLC [Novapak C-18; 5% acetonitrile]. The fractionscontaining >90% 2-CdA were pooled and concentrated to dryness. The crudeproduct residue from the first Dowex column was dissolved in water andloaded on a second polystyrene anion exchange resin (Dowex 1×4 200-400mesh, 2000 ml) column. This second Dowex column was eluted with wateruntil all early running impurities were removed (˜14 L). The column wasthen eluted with 10% MeOH/water and the eluate collected from the columnin fractions was analyzed by HPLC. The fractions containing >99% 2-CdAwere pooled and concentrated to a small volume. The concentrated poolwas stored overnight at 5° C. to allow crystallization. The crystalswere filtered, washed with water and dried under reduced pressure. Theyield of the product (99% by HPLC) is 27% (17.4 g). This isapproximately ten fold (1000%) greater than the methods reported in U.S.Pat. No. 5,208,327.

The identity of the product was established by using well knownanalytical methods. The analytical data is listed below: UV max 294 nm,ε14749 (0.1 mg/mL in 0.1M NaOH aq); MS FAB MS, m/z 286.1 [M+H]+, HR FABMS [M+Na]+ Expected 308.05267; Found 308.0522; NMR 1H (, ppm) 8.4 (1H),7.8 (2H), 6.3 (1H), 5.3 (1H), 4.9(1H), 4.4 (1H), 3.9 (1H), 3.6 (1H), 3.3(2H), 2.6 (1H), 2.3 (1H); 13C (, ppm) 156.721, 152.889, 150.011,139.736, 118.117, 97.892, 83.512, 70.653, 61.607, 39.8 (buried in DMSOsolvent resonance); Reverse Phase HPLC-Novapak-C-18 column, 5%acetonitrile/water, 0.1 M TEA pH 7 [with acetic acid] buffer retentiontime 10.56 min.; Elemental Analysis: Calculated %C 42.04%H 4.23%N24.51%Cl 12.41; Found %C 41.71%H 4.33%N 24.31%Cl 12.78

EXAMPLE 3 Synthesis of 2-chloro-2′-deoxyadenosine using CuCl₂ as MetalHalide Lewis Acid

DAPD (5 g, 18.8 mmol) is suspended in 425 mL dichloroethane/DMSO (4:1)and cooled to 0° C. under an inert atmosphere. To this is added Copper(II) chloride (9 g, 66.9 mmol) and tert-butyl nitrite (4.6 ml, 4.0 g,38.6 mmol) and stirred while allowing the reaction to warm to roomtemperature. The reaction was stirred for 4 days and quenched withtriethylamine. The reaction mixture was evaporated to dryness and theresidue dissolved in sodium bicarbonate solution and purified by columnchromatography. The product containing fractions were pooled togetherand evaporated to dryness the solid was recrystallized from water togive crystalline product. The overall yield of 2-CdA from startingmaterial is 20% (0.9 g). The product was identified by HPLC [NovapakC-18; 5% acetonitrile/water, 0.1M TEA, pH 7 with acetic acid]; itcomigrated with a coinjected sample of 2-CdA. This reaction establishesthat metal halides of various compositions, so long as they are Lewisacids, work in the methods of the present invention.

EXAMPLE 4 Synthesis of 2-chloro-2′-deoxyadenosine using Pentyl Nitrite

DAPD (1 g, 0.375 mol) was suspended in dichloroethane (70 mL)/DMSO (15mL) (˜4:1) and cooled to less than 10° C. under an inert atmosphere. Tothis was added antimony trichloride (1.2 g, 0. mol) and pentyl nitrite(2.73 g, 3.1 mL, 0.0233 mol) and stirred while allowing the reaction towarm to room temperature. After 4 hrs the reaction was 19.2% complete bypeak area on HPLC [Phenosphere 5 μM ODS2; 8% acetonitrile/water, 0.025MKH₂PO₄, pH 3]. An additonal 4 equivalents of pentyl nitrite (1.82 g,2.06 mL, 0.0155 mol) was added. The reaction was stirred overnight andwas quenched with triethylamine. The overall conversion to product was31% by HPLC. The reaction product was confirmed by HPLC by coinjectionwith a standard sample.

EXAMPLE 5 Synthesis of 2-chloro-2′-deoxyadenosine indichloroethane/dimethylformamide

DAPD (2 g, 7.5 mmol) was suspended in 170 mL dichloroethane/DMF (4:1)and cooled to 0° C. under an inert atmosphere. To this was addedantimony trichloride (3.8 g, 10.5 mmol) and tert-butyl nitrite (1.85 ml,1.6 g, 15.5 mmol) and stirred while allowing the reaction to warm toroom temperature. Analysis of the reaction mixture by HPLC [NovapakC-18; 5% acetonitrile/water, 0.1M TEA, pH 7 with acetic acid] revealedproduct formation, confirmed by comigration with a coinjected 2-CdAstandard. This experiment establishes that polar aprotic solvents otherthan DMSO can be used in the methods of the invention.

EXAMPLE 6 Synthesis of 2-chloro-2′-deoxyadenosine inDichloromethane/DMSO

DAPD (2 g, 7.5 mmol) was suspended in 170 mL dichloromethane (DCM)/DMSO(4:1) and cooled to 0° C. under an inert atmosphere. To this was addedantimony trichloride (3.8 g, 10.5 mmol) and tert-butyl nitrite (1.85 ml,1.6 g, 15.5 mmol) while stirring while the reaction was allowed to warmto room temperature. Analysis by HPLC (Novapak C-18; 5%acetonitrile/water, 0.1M TEA, pH 7 with acetic acid) revealed productformation, which was confirmed by comigration of a product peak with acoinjected 2-CdA standard.

EXAMPLE 7 Determination of Optimal Solvent Combination

The syntheses were performed as in Example 1, except that the ratio ofthe two solvents was varied and the reactions were periodicallymonitored by HPLC [Novapak C-18; 5% acetonitrile/water, 0.1M TEA, pH 7with acetic acid]. The results are presented in Table 1, below. The timevs. concentration of product 2-CdA and starting material DAPD suggestedthat a 1:10 to 10:1 ratio of the polar aprotic to nonpolar chlorinatedis optimal for these reactions, with a 1:4 ratio being most optimal.

DMSO/ Time of Reaction (hours) DCE Ratio 16 22 40 72 120 100/0  17.215.9 10/1  20.1 23.3 19.2 1/1 30.4 36.7 35 36.3 1/4 18.3 57.3  1/10 27.725.9 14.4

EXAMPLE 8 Synthesis of 2-chloroadenosine

Diaminopurine ribonucleoside (“DAPR”) (1.0 g, 3.54 mmol) was suspendedin 80.0 mL dichloroethane/DMSO (4:1) and was cooled to less than 10° C.under an inert atmosphere. To this was added antimony trichloride (1.14g, 5.01 mmol) and tert-butyl nitrite (0.872 ml, 7.4 mmol) and stirredwhile allowing the reaction to warm to room temperature. Analysis byHPLC (Phenosphere 5 μM ODS2; 8% acetonitrile/water, 0.025M KH₂PO₄, pH 3)showed a new peak around 15 min with concomitant loss of startingmaterial DAPR. The reaction was stirred overnight and was quenched withtriethylamine. The total conversion by HPLC was 23%. The product wasidentified by coinjection with pure 2-chloroadenosine obtained fromSigma Chemical Company, St. Louis, Mo.

EXAMPLE 9 Synthesis of 2-chloroadenine

Diaminopurine Hydrochloride [DAP] (2 g, 7.5 mmol) was suspended in 170mL dichloroethane/DMSO (4:1) and cooled to 0° C. under an inertatmosphere. To this was added antimony tribromide (3.8 g, 10.5 mmol) andtert-butyl nitrite (1.85 mL, 1.6 g, 15.5 mmol) followed by stirringwhile allowing the reaction to warm to room temperature. Analysis byHPLC [Novapak C-18; 5% acetonitrile/water, 0.1M TEA, pH 7 with aceticacid] revealed a new peak that eluted at 5.59 minutes with concomitantloss of DAP starting material. The reaction was stirred overnight andquenched with triethylamine. HPLC showed new peak indicating thesynthesis of 2-chloroadenine.

A sample of 2-chloro-2′-deoxyadenosine was taken and subjected towell-established deglycosylation conditions to give a mixture of both2-chloroadenine and 2-chloro-2′-deoxyadenosine. This sample whenco-injected with the reaction mixture from the previous Example showedoverlapping peaks at 5.59 min verifying the formation of2-chloroadenine. The product, 2-chloroadenine also appears at 8.78 minby HPLC in the Phenosphere 5 μM ODS2; 8% acetonitrile/water, 0.025MKH₂PO₄, pH 3 system.

This Example, along with the other Examples set forth herein, establishthe generality of the halogenation reaction of the invention, such thatany substituted or unsubstituted 2-halo-6-aminopurine can be producedfrom the corresponding substituted or unsubstituted 2,6-diaminopurine.

EXAMPLE 10 Synthesis of 2-bromo-2′-deoxyadenosine

DAPD (2 g, 7.5 mmol) was suspended in 170 mL dichloroethane/DMSO (4:1)and cooled to 0° C. under an inert atmosphere. To this was addedantimony tribromide (3.8 g, 10.5 mmol) and tert-butyl nitrite (1.85 mL,1.6 g, 15.5 mmol) followed by stirring while allowing the reaction towarm to room temperature. Analysis by HPLC [Novapak C-18; 5%acetonitrile/water, 0.1M TEA, pH 7 with acetic acid] revealed a new peakthat eluted at 5.59 minutes (a retention time that was expected for thebrominated product) with concomitant loss of DAPD starting material. Thereaction was stirred overnight and quenched with triethylamine. Theconversion by HPLC was 26% indicating the synthesis of2-bromo-2′-deoxyadenosine.

EXAMPLE 11 Synthesis of 2-fluoro-2′deoxyadenosine

DAPD (2 g, 7.5 mmol) was suspended in 170 mL dichloroethane/DMSO (4:1)and cooled to 0° C. under an inert atmosphere. To this is added antimonytrifluoride (1.88 g, 10.5 mmol) and tert-butyl nitrite (1.85 ml, 1.6 g,15.5 mmol) followed by stirring while allowing the reaction to warm toroom temperature. Analysis of the reaction mixture by HPLC [NovapakC-18; 5% acetonitrile/water, 0.1M TEA, pH 7 with acetic acid] revealed anew peak which eluted at 6.4 min, indicating the synthesis of2-fluoro-2′-deoxyadenosine with concomitant loss of starting material.The reaction was stirred overnight and quenched with triethylamine. Theconversion to product by HPLC was 8%.

The examples 1, 10 and 11 show the generality of the halogenationreaction by metal halides, such that any 2-halo substituted purinenucleosides can be produced from the corresponding 2,6-diaminonucleoside.

EXAMPLE 12 Synthesis of 2-chloro-2′-O-methyl-adenosine

2′-OMe-diaminopurine ribonucleoside (2 g, 6.7 mmol) was suspended in 152ml dichloroethane/DMSO (4:1) and cooled to less than 10° C. under aninert atmosphere. To this was added antimony trichloride (2.14 g, 9.4mmol) and tert-butyl nitrite (1.65 ml, 1.4 g, 14 mmol) followed bystirring while allowing the reaction to warm to room temperature.Analysis of the product mixture by HPLC [Novapak C-18; 5%acetonitrile/water, 0.1M TEA, pH 7 with acetic acid] revealed a new peakthat eluted at 11.7 min with concomitant loss of starting material. Thereaction was stirred overnight and quenched with triethylamine. Theappearance of the new peak (51%), and disappearance of startingmaterial, indicates the synthesis of 2-chloro-2′-O-methyl-adenosine, andestablishes the generality of the method to 2′-substituted nucleosidesas well as 2′-deoxynucleosides.

EXAMPLE 13 Preferred Stabilazation and Purification

A. Stabilization Column (Amberchrom Chromatography)

An Amberchrom CG161 XUS resin column (15 L) was prepared (˜4 g 2-CldAper L of resin). Two-thirds of the resin was removed from the column andslurried with the reaction mixture and was then loaded onto the column.The column was washed with water until the absorbance at 265 nm dropsbelow 5 or until the 2-CldA began to elute off the column and then 3 to6 L fractions were collected. This was followed by elution with 5%methanol in water, and then with 30% methanol in water until theabsorbance of the eluate at 265 nm was below 10. All fractionscontaining greater than 75% 2Cl-dA by peak area using HPLC werecollected and concentrated on a rotary evaporator at 45° C. Thecollected fractions were concentrated to a gum, which was in a stablestate and which could be left for up to 7 days without degrading.

B. Purification Column 1(Dowex Chromatography)

The gum from step A was dissolved in water and then concentrated to aconcentration of 12-15 g of 2-CldA/L. This solution was then chargedonto a Dowex 1×4 anion exchange resin (1.3 L) column. The column waseluted first with water until the absorbance of the eluate at 265 nm wasbelow 10, followed by 10% methanol in water, 15% methanol in water, andfinally 25% methanol in water until all the 2-CldA had eluted off thecolumn. Fractions containing 2-CldA were collected and assayed forpurity by HPLC. The fractions that contained >90% pure 2Cl-dA (by HPLC)were adjusted to pH 7.5 or greater by using 1.0M sodium bicarbonatesolution to achieve a concentration 10 mM sodium bicarbonate. These weresaved individually, without pooling.

C. Purification Column 2 (Dowex Chromatography)

A second Dowex 1×4 anion exchange resin column (6 L) was prepared. (1 Lof resin per 10 g of 2-CldA). The column was equilibrated in 10 mMsodium bicarbonate solution before the charge of 2-CldA was loaded. Thecharge was made up of the fractions from the 1×4 column in Step B. Thefractions were loaded onto this 1×4 column in the order that they elutedoff the first 1×4 column. After the column was loaded, the first eluant,10 mM sodium bicarbonate in water, was started over the column. Once themajority of the 2-CldA was eluted off the column, the buffer wasswitched to 10% methanol in a 10 mM sodium bicarbonate solution. Thecolumn was washed with this eluant until 90% or more of 2Cl-dA hadeluted off the column. All fractions that contained 2-CldA at greaterthan 98% purity by HPLC were concentrated in a rotary evaporator at 45°C. under vacuum.

D. Crystallization

The main (>98% pure) product pool was evaporated to a concentration of22 g of 2-CldA per liter. The concentrated pool was filtered using aBuchner funnel and Whatman 54 hardened filter paper. The filteredsolution was sealed and placed in a refrigerator for 17 hours at 8-14°C. for crystallization. The concentrated solution should remain at 8-14°C. for at least 14 hours. The crystallized 2-CldA was collected byfiltration. The solids were washed with cold water. Both the solids andthe filtrate were assayed for 2-CldA using HPLC.

E. Recrystallization 1

The solids from crystallization step D were redissolved in 10 mM sodiumbicarbonate solution at approximately 4-5 grams of 2-CldA per L and werethen concentrated down to 22 g/L using a rotary evaporator. Theconcentrated solution was then filtered and placed in the refrigeratorfor 17 hours for recrystallization. After 17 hours, the solids werefiltered and both the solids and filtrate are assayed by HPLC.

F. Recrystallization 2

A significant amount of 2-CldA was detected in the filtrate from therecrystallization 1, step E, which was greater than 99% purity by HPLC.The filtrate was concentrated and recrystallized to recover 2-CldA. Allthe product that passed the 99% purity and the <0.1% impurityspecifications were placed in a vacuum oven at a temperature of 37° C.for 24 hours to dry. After drying the crystals were ground up and drieduntil the water content was 2% or less. The crystals were weighed for afinal yield.

For maximum recovery, fractions with <98% pure 2-CldA obtained from theDowex Column 2, step C above, were pooled and reprocessed over a 1×4Dowex column, using the same procedure as described in step C. The clean[>98%] fractions were combined with the filtrates from therecrystallizations. This solution was then concentrated andrecrystallized as described above in step E.

What is claimed is:
 1. A method for producing2-halo-6-aminopurine-2′,3′-derivatized nucleosides comprising the stepsof: admixing a nonpolar aprotic organic solvent with a polar aproticorganic solvent to produce a solvent mixture; dissolving in said solventmixture a nucleoside having the formula

 wherein Q is S; wherein R¹ and R² are a moiety with the formulaO—A(Y)—O, wherein A is C, S, or P—R and wherein Y is O, S. N—R, or 2R;or wherein R¹ and R² are independently hydrogen, O—R, R, N—R₂, N₃, X, orS—R; wherein R is a linear or branched chain alkyl, cycloalkyl,alkoxyalkyl, ether, thioether, haloalkyl, aryl group, a monocyclic arylgroup, a multicyclic aryl group, or a heterocyclic aryl group havingfrom 1 to 20 carbon atoms and 1 to 10 heteroatoms, and X is Cl, Br, F,or I; reacting said nucleoside in said solvent mixture with an organicnitrite and a metal halide, wherein said metal halide is a Lewis acid,to produce a reaction product.
 2. A method for producing2-halo-6-aminopurine-2′-deoxy or 2′-substituted N-7 glycosylatednucleosides comprising the steps of: admixing a nonpolar aprotic organicsolvent with a polar aprotic organic solvent to produce a solventmixture; dissolving in said solvent mixture an unprotected N-7glycosylated nucleoside having the formula

 wherein Q is O or S; wherein R¹ and R² are a moiety with the formulaO—A(Y)—O, wherein A is C, S, or P—R and wherein Y is O, S, N—R, or 2R;or wherein R¹ and R² are independently hydrogen, O—R, R, N—R₂, N₃, X, orS—R; wherein R is linear or branched chain alkyl, cycloalkyl,alkoxyalkyl, ether, thioether, haloalkyl, a monocyclic aryl group, amulticyclic aryl group, or a heterocyclic aryl group having from 1 to 20carbon atoms and 1 to 10 heteroatoms, and X is Cl, Br, F, or I; andreacting said unprotected nucleoside in said solvent mixture with anorganic nitrite and a metal halide, wherein said metal halide is a Lewisacid, to produce a reaction product.